Significance of Sample Preprocessing
In metabolomics research, sample preprocessing is a critical phase in the sample preparation process. Its primary objective is to extract metabolites from biological samples maximally while eliminating potential interference factors that could affect experimental results. Common sample preprocessing steps include sample collection, storage, extraction, and derivatization.
Firstly, meticulous consideration during sample collection and preservation is vital for the reliability of metabolomics data. Variations in metabolite content and composition may exist among different biological tissues, making careful sample collection a prerequisite for ensuring data reliability. Additionally, proper sample storage conditions, such as low-temperature freezing or the addition of preservatives, can effectively prevent the degradation of metabolites within the sample.
Secondly, the extraction process directly influences the sensitivity and coverage of metabolite detection. Common extraction methods include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The choice of extraction method depends on the specific goals of the study and the nature of the samples.
Lastly, sample derivatization is a key step in enhancing the sensitivity of metabolite detection. Derivatization alters the properties of metabolites, making them more easily detectable by mass spectrometry. The optimization of this step is crucial for the detection of low-abundance metabolites.
The choice of sample pre-treatment strategy contributes to the success of a given experiment as it affects the quality of the observed metabolite profiles and data. The capabilities and limitations of the sample preparation method in a given study can affect the accuracy of the biological interpretation.
Ideal state of sample pre-processing
The ideal state of metabolomics sample preprocessing must fulfill the following conditions: 1) non-selective; 2) few and fast steps; 3) reproducible; and 4) include metabolic termination processes. So far there is no single pre-treatment that can fulfill all of these requirements.
From the perspective of biomarker discovery, one of the most contradictory demands is the termination of metabolism, encompassing samples from cells, plants, and tissues. The purpose of metabolic termination is to halt the metabolic processes using low temperature, acidification, or rapid heating. However, metabolic processes are extremely rapid, often occurring on a timescale of less than 1 second, as seen with molecules like ATP, 6-phospho-glucose, and adenosine. Therefore, implementing appropriate termination steps within the appropriate timescale can be challenging, and adding an extra termination step may unintentionally lead to degradation or loss of certain metabolites.
For instance, in a study by Deprez S involving rat plasma stored at low temperatures, residual enzymes caused an increase in levels of choline, glycerol, tyrosine, and phenylalanine. For stable metabolites, the significance of metabolic termination is minimal. However, for unstable metabolites prone to degradation or conversion, the termination step is crucial. Though these unstable metabolites may constitute a small proportion, they could be essential biomarkers; for example, adenosine and glutathione, mentioned earlier, have been reported as biomarkers for cancer.
Therefore, determining which metabolites are unaffected by different termination/storage conditions plays a crucial role in the maturation of the field of metabolomics and the discovery of biomarkers.
Preprocessing Methods for Biological Fluid Samples
Direct Dilution Injection
The direct dilution injection method is a widely utilized approach specifically tailored for urine samples. In this technique, a standard dilution ratio of 1:10 is employed, utilizing pure water for the dilution process. This straightforward yet effective method ensures that the biological fluid samples, particularly urine and plasma, maintain their integrity throughout the LC-MS analysis. Figure 1 provides a visual representation of the recommended workflow, highlighting the simplicity and efficiency of this method in metabolite analysis.
Figure 1. Recommended LC-MS Analytical Procedures for Biological Fluids such as Urine and Plasma
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Solvent Precipitation
Solvent precipitation is a crucial technique applied to biological fluid samples, notably serum and plasma. By introducing organic solvents such as methanol, acetonitrile, ethanol, acetone, or combinations thereof, this method aims to selectively remove proteins. The strategic addition of these solvents disrupts the cross-linking interactions between proteins and metabolites. The resulting concentration of metabolites obtained through solvent precipitation serves as a representative measure of the total metabolite concentration within the sample.
Ultrafiltration
Ultrafiltration emerges as a prevalent and molecular weight-based separation technique in the realm of biological sample preparation. A membrane with a specified cutoff, for instance, 3000 Da, is employed to effectively segregate small-molecule metabolites from larger proteins or molecules. It is essential to recognize that ultrafiltration exhibits a distinct bias toward polar molecules. However, compared to solvent precipitation, it may lead to a notable loss of hydrophobic metabolites. Despite this, ultrafiltration remains a valuable method for precise molecular separation in metabolomics studies.
Solid-Phase Extraction (SPE)
SPE is a versatile and widely adopted method in targeted biological analysis. This method involves a systematic approach: firstly, analytes adsorb onto the surface of a solvent; secondly, interfering substances with weaker adsorption capacity are removed through a washing process; finally, elution of analytes occurs using a solvent. Notably, the integration of well-known materials such as the C18 column with polystyrene-divinylbenzene adsorbents has expanded the application of SPE to non-targeted metabolomics research. The current challenge in SPE lies in the formulation of universally applicable extraction conditions that can accommodate the diverse nature of metabolites present in biological samples.
Development Trends in Biological Sample Preprocessing
In vivo Sampling: Solid-Phase Microextraction (SPME)
In vivo sampling avoids potential changes in the metabolic profile caused by exposure to oxygen, solvents, and pH conditions during the sampling process, as well as the activation of various biological processes. SPME technology provides a balanced extraction of hydrophilic and hydrophobic metabolites and can be applied to in vivo sampling of circulating blood metabolites in mice.
Turbulent Flow Chromatography (TFC)
TFC allows the direct injection of untreated serum into LC-MS, as it meets the conditions for both large particles (25-50 μm) and small particle sizes (0.5-1.0 μm). Large molecular substances are not retained, while small molecules can be detected. The potential advantages of TFC include high throughput, high automation, and minimal sample processing, which can reduce the introduction of external contaminants and unintended sample loss in the sample processing workflow.
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